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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.80, #2)
Hyperproduction of l-glutamate oxidase in submerged fermentation of Streptomyces sp. N1 with culture pH control and calcium addition by Qing-Shan Li; Qing-Hua Fang; Jian-Yun Zhu; Jian-Jiang Zhong (pp. 97-106).
Production of l-glutamate oxidase (GluOx) by Streptomyces sp. N1 was investigated by controlling culture pH at 6.2, 6.7, 7.0, and 7.3 in a 5-l stirred fermentor. The corresponding GluOx activities obtained were 2.8, 4.2, 6.0, and 5.3 U/mL, respectively. Microbial growth was inhibited by increasing the medium pH from 6.2 to 7.0. The inhibitory effect was also observed in plate colony growth under incubation with a different initial pH value. The effect of calcium on GluOx production was also studied in the pH-controlled bioreactor. When the culture pH was controlled at 6.2 or 7.0, GluOx production could not be improved or was only improved slightly by initial addition of calcium to the medium. However, when the culture pH was kept at 6.7, initial Ca2+ addition (60 mM) conspicuously enhanced GluOx production up to 9.3 U/mL, which was about twofold of that without Ca2+ addition. The enzyme production level was the highest ever reported in the literature. During fermentation the inhibition of cell growth by Ca2+ addition was observed. For the morphological changes, the cells mostly existed as pellets in the medium without Ca2+ addition, whereas few pellets were found and almost all the cells were dispersed mycelia in the broth with Ca2+ addition.
Keywords: l-Glutamate oxidase; submerged fermentation; calcium addition; pH control; morphological change; Streptomyces sp
Dissolved oxygen concentration affects the accumulation of HIV-1 recombinant proteins in Escherichia coli by M. Walid Qoronfleh (pp. 107-120).
A central problem in aerobic growth of any culture is the maintenance of dissolved oxygen concentration (DOC) above growth-limiting levels especially in high-cell density fermentations that are usually of the fed-batch type. Fermentor studies have been conducted to determine the influence of DOC on the production of heterologous proteins in Escherichia coli. The results demonstrated that there is a significant degree of product-to-product variation in the response of heterologous protein accumulation to DOC. For translational fusions of the human immunodeficiency virus-1 (HIV-1) proteins p24Gag and Env41, the imposition of a dissolved oxygen (DO) limitation resulted in 100 and 15% increases in the respective product yields. On the other hand, the imposition of a DO limitation had no effect on the production of a similar translational fusion of the HIV-1 protein p55Gag, and a large negative effect on the production of an influenza protein (C13). The stimulatory effects of DOC on p24Gag production were investigated further. The results of my studies suggested that the stimulatory effect observed at reduced agitation rates on p24Gag accumulation was owing to an oxygen effect and not a shear effect. Furthermore, the results of my investigations indicated that the effect a DOC had on the production of p24Gag was strongly influenced by the cell density at which the culture was induced.
Keywords: Dissolved oxygen; HIV-1 proteins; recombinant proteins; Escherichia coli ; heterologous gene expression
Molecular cloning and expression of an endo-β-1,4-d-glucanase I (avicelase I) gene from Bacillus cellulyticus K-12 and characterization of the recombinant enzyme by Tae-Kyun Lee; Cheorl-Ho Kim (pp. 121-140).
Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an M r , of 64,000 was detected. The recombinant endo 1,4-β- d -glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. K m values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas V max values were 201 and 9.2 μmol · min−1 · mg−1, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg++ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.
Keywords: Gene cloning; expression; avicelase; endo-β-1,4-d-glucanase; cellobiohydrolase; xylanase; Bacillus cellulyticus K-12
Eicosapentaenoic acid release from the red alga Pachymeniopsis lanceolata by enzymatic degradation by Ritsuko Matsukawa; Kiyotaka Hatakeda; Shota Ito; Yukiyo Numata; Hidetoshi Nakamachi; Yasushi Hasebe; Shunichi Uchiyama; Masahiro Notoya; Zvy Dubinsky; Isao Karube (pp. 141-150).
Forty-eight species of seaweeds from Japanese waters were screened for the valuable polyunsaturated fatty acids eicosapentaenoic acid (EPA). The eight species that contained the highest levels of these compounds were analyzed in detail. Of all species tested the red alga Pachymeniopsis lanceolata contained the highest EPA concentration, and it was present as both the free and bound forms. EPA constituted 38.7% of total fatty acids, and polar lipids were the main constituent of the total lipids in P. lanceolata. EPA was obtained from the marine algae P. lanceolata by enzymatic hydrolysis of the total lipids extract using phospholipase A2(PLA2). The release of EPA reached a plateau after 10 min of enzymatic treatment. These results suggest that P. lanceolata is a useful natural source of EPA and that PLA2 treatment is a convenient method for obtaining EPA from the red alga.
Keywords: Eicosapentaenoic acid; phospholipase A2 ; enzymatic hydrolysis; seaweeds; Pachymeniopsis lanceolata
Experimental and modeling studies of diffusion in immobilized cell systems
by Mark R. Riley; Fernando J. Muzzio; Sebastian C. Reyes (pp. 151-188).